The type III secretion system (TTSS) is essential to virulence in a large group of medically important Gram-negative bacterial pathogens, including Yersinia spp., Salmonella spp., and Escherichia coli O157:H7 among others. The TTSS exerts its role in virulence through translocation of bacterial proteins termed effectors into the interior of host cells. Most effectors are crucial to virulence and have deleterious consequences in host cells. Our long-term goal is to understand the steps required for transport of proteins by the TTSS. This proposal focuses on TTSS chaperone proteins. TTSS chaperones form a large and distinctive family of functionally important and conserved proteins. Members of this family have been found to be required for translocation of corresponding effector proteins into host cells, explaining why chaperones are as crucial to virulence as their corresponding effectors. Recent structural and biochemical advances suggest that TTSS chaperones have a conserved mechanism of action in promoting translocation of effectors. However, this process is still not understood in detail. This proposal seeks to build on these recent structural and biochemical advances to determine the mechanism of action of TTSS chaperones. Our specific aims are focused on the well characterized Yersinia effector YopE and its corresponding chaperone SycE. Our first aim is to determine directly whether SycE promotes localized YopE unfolding for transport through the narrow needle-like TTSS apparatus. Our second aim is to map functionally important sites on the surface of the SycE-YopE complex, which are likely to be responsible for conferring association with bacterial components involved in the TTSS process, such as dissociation factors. Our third aim is to identify bacterial components that associate with SycE-YopE and to understand the functional consequence of association. These aims are synergistic, in that structurally altered or functionally important surface regions of SycE-YopE, as identified by the first two aims, respectively, will be understood in the context of interacting bacterial components, as identified by the third aim. The combination of these three aims is likely to enable mechanistic dissection of steps required for TTSS transport of proteins. This multidisciplinary approach, which combines structure, genetics, and biochemistry, is likely to be valuable in the design of antimicrobial strategies aimed at combating the large class of bacterial pathogens that use the TTSS in virulence. A large number of bacterial pathogens use a needle-like apparatus to inject bacterial proteins, which are often toxic, into human cells. Our proposal is aimed at understanding how this injection process occurs, and results from our studies are likely to be valuable in the design of antimicrobial strategies aimed at combating these pathogens.